The a-factor pheromone of Saccharomyces cerevisiae is secreted by a cells and mediates mating between a and alpha cells. A combined biochemical, genetic, and molecular approach will be used to study the biogenesis of a-factor and determine its roles in mating. Biogenesis of a-factor. Mature, secreted a-factor is twelve residues long and contains a covalent modification, likely a lipid. It is derived by proteolytic processing from precursors encoded by the MFAl and MFA2 genes. The lack of a signal sequence in these precursors suggests that a-factor export involves a novel secretory pathway. The post-translational reactions required for a-factor maturation (proteolytic processing, modification, and secretion) will be analyzed by: a) Using immunochemical methods to directly identify a-factor biosynthetic intermediates in wild-type strains and in mutants (ste6, ste14, and ram) in which a-factor production is blocked. b) Determining the sites within a-factor which are critical for each step in maturation by mutagenesis of MFAl and analysis of the fate of the mutant products. c) Studying the protein components which mediate these reactions by a molecular analysis of the STE6, STE14, and RAM genes. Roles of a-factor in mating. While it has been established that the secreted form of a-factor triggers preparatory mating responses (such as cell cycle arrest) in alpha partners, it is likely that a cell-associated form of a-factor plays a role in other steps in mating. By mutational analysis of MFAl, regions of_a-factor which are required for these distinct activities will be identified. In addition, an alpha cell mutant defective in a proposed a-factor inactivating activity will be sought. The study of a-factor biogenesis and function will provide information about a variety of fundamental biological processes including how proteins are shaped by proteolytic processing and covalent modification, how molecules are secreted from cell and how cells communicate with one another.